Choline kinase (also known as CK, CHK and ChoK) is the initial enzyme of the Kennedy or phosphatidylcholine (PC) synthesis pathway and phosphorylates choline to phosphorylcholine (PCho) in the presence of magnesium (M.g.sup.2+) using adenosine 5′-triphosphate (ATP) as a phosphate group donor. The transformation mediated by various oncogenes induces high levels of choline kinase activity, giving rise to an abnormal increase in the intracellular levels of its product, PCho, which indirectly supports the role of choline kinase in generating human tumors. However, there are alternative PCho generation mechanisms that do not involve the activation of choline kinase and could explain the high levels of this metabolite in tumor cells.
Although there is evidence of increase in activity of the enzyme choline kinase in tumors and transformed cells, its relationship to the carcinogenic process is not sufficiently demonstrated as no clear cause-effect relationship has been established between the increase in activity and the tumor transformation. On the other hand, the molecule responsible for this effect has still not been identified.
About 200 gene sequences encoding for polypeptides with a primary structure homologous to choline kinase have been identified and are designated as choline kinase alpha a, choline kinase alpha b, choline kinase alpha 3, choline kinase beta 1, choline kinase beta 2, choline kinase CKB-1 Choline/ethanolamine kinase, choline kinase-like ethanolamine kinase, Cots, Duff227, Cog3173 CPTIB, SFI, SHOX2, FHOD2, FLJ12242, KRT5, FBL, ARL6IP4, etc. both in humans and in other mammals and rodents (rats, mice, cows, guinea pigs, rabbits, monkeys). In fact, since 1982 there has been biochemical evidence that in different tissues isolated from rats, mice and humans there are at least three isoenzymes with choline kinase activity showing different physicochemical properties.
At least 3 genes encoding for proteins with demonstrated choline kinase activity have recently been identified in human genoma, designated as ck-alpha, ck-beta, and HCEKV (USA patent US2003186241), and several genes the encoded proteins of which are 30-65% homologous to those encoded by the ck genes, such as for example the genes CAI16602, CHKL, CAI16600, CAI16599, CAH56371, CAI16603, BAA91793, and CAI16598, and the genes CPTIB, EKI2, SFI, SHOX2, FHOD2, FLJ12242, KRT5, FBL, ARI61p$, TOMM40, and MLL. A very relevant characteristic of the different choline kinase isoenzymes is that they have different biochemical properties, with important variations in their affinity for the choline substrate or for the ATP phosphate donor, and even in their active form, which can be presented as dimers or tetramers. Therefore it is necessary to define if there is a direct relationship between any of the different choline kinase isoenzymes identified and the attributed tumorigenic capacity due to their overexpression in human tumors.
On the other hand, choline kinase inhibition has been demonstrated to be a new and effective anti-tumor strategy in cells transformed by oncogenes, which has been extrapolated to nude mice in vivo. The increase in choline kinase activity in several human breast carcinomas has recently been published, and it has been seen that the choline kinase alteration is a frequent event in some human tumors such as lung, colorectal and prostate tumors.
Despite the correlation between some parameters and others, there is currently no evidence definitely establishing that the overexpression of choline kinase has oncogenic and tumor activity in human cells. There is evidence indicating that choline kinase activity inhibitors, such as hemicholinium-3 [Cuadrado A., Carnero A., Dolfi F., Jimenez B. and Lacal J. C. Oncogene 8, 2959-2968 (1993); Jimenez B., del Peso L., Montaner S., Esteve P. and Lacal J. C. J. Cell Biochem. 57, 141-149 (1995); Hernandez-Alcoceba, R., Saniger, L., Campos, J., N nez, M. C., Khaless, F., Gallo, M., Espinosa, A, Lacal, J. C. Oncogene, 15, 2289-2301 (1997)] or the low-toxicity methylenequinones in Spanish patent application ES200503263, present anti-tumor activity. However, there is no conclusive evidence in the mentioned documents or in the rest of the prior art as regards to the various isoenzymes with demonstrated choline kinase activity (ck-alpha, ck-beta, HCEKV, etc) and identified in human tissues could be responsible for the detected enzymatic activity, nor is it indicated which of the isoenzymes is sensitive to the inhibition by inhibitors which have shown anti-tumor activity. This identification is necessary in order to be able to establish its potential use as a therapeutic target in cancer.